Alzheimer's disease (AD) affects more than 10% of the population ≥65 y of age, but the underlying biological risks of most AD cases are unclear. We show anti-poly-glycine-arginine (a-polyGR) positive aggregates frequently accumulate in sporadic AD autopsy brains (45/80 cases). We hypothesize that these aggregates are caused by one or more polyGR-encoding repeat expansion mutations. We developed a CRISPR/deactivated-Cas9 enrichment strategy to identify candidate GR-encoding repeat expansion mutations directly from genomic DNA isolated from a-polyGR(+) AD cases. Using this approach, we isolated an interrupted (GGGAGA)n intronic expansion within a SINE-VNTR-Alu element in CASP8 (CASP8-GGGAGAEXP). Immunostaining using a-polyGR and locus-specific C-terminal antibodies demonstrate that the CASP8-GGGAGAEXP expresses hybrid poly(GR)n(GE)n(RE)n proteins that accumulate in CASP8-GGGAGAEXP(+) AD brains. In cells, expression of CASP8-GGGAGAEXP minigenes leads to increased p-Tau (Ser202/Thr205) levels. Consistent with other types of repeat-associated non-AUG (RAN) proteins, poly(GR)n(GE)n(RE)n protein levels are increased by stress. Additionally, levels of these stress-induced proteins are reduced by metformin. Association studies show specific aggregate promoting interrupted CASP8-GGGAGAEXP sequence variants found in ~3.6% of controls and 7.5% AD cases increase AD risk [CASP8-GGGAGA-AD-R1; OR 2.2, 95% CI (1.5185 to 3.1896), P = 3.1 * 10-5]. Cells transfected with a high-risk CASP8-GGGAGA-AD-R1 variant show increased toxicity and increased levels of poly(GR)n(GE)n(RE)n aggregates. Taken together, these data identify polyGR(+) aggregates as a frequent and unexpected type of brain pathology in AD and CASP8-GGGAGA-AD-R1 alleles as a relatively common AD risk factor. Taken together, these data support a model in which CASP8-GGGAGAEXP alleles combined with stress increase AD risk.

Microsatellite-expansion mutations cause >50 neurological diseases but there are no effective treatments. Mechanistic studies have historically focused on protein loss-of-function and protein or RNA gain-of-function effects. It is now clear that many expansion mutations are bidirectionally transcribed producing two toxic expansion RNAs, which can produce up to six mutant proteins by repeat associated non-AUG (RAN) translation. Multiple types of RAN proteins have been shown to be toxic in cell and animal models, to lead to common types of neuropathological changes, and to dysregulate key pathways. How RAN proteins are produced without the canonical AUG or close-cognate AUG-like initiation codons is not yet completely understood but RNA structure, flanking sequences and stress pathways have been shown to be important. Here, we summarize recent progress in understanding the role of RAN proteins, mechanistic insights into their production, and the identification of novel therapeutic strategies that may be applicable across these neurodegenerative disorders.

Purpose of review An intronic G(4)C(2) expansion mutation in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). Although there are currently no treatments for this insidious, fatal disease, intense research has led to promising therapeutic strategies, which will be discussed here. Recent findings Therapeutic strategies for C9-ALS/FTD have primarily focused on reducing the toxic effects of mutant expansion RNAs or the dipeptide repeat proteins (DPRs). The pathogenic effects of G(4)C(2) expansion transcripts have been targeted using approaches aimed at promoting their degradation, inhibiting nuclear export or silencing transcription. Other promising strategies include immunotherapy to reduce the DPRs themselves, reducing RAN translation, removing the repeats using DNA or RNA editing and manipulation of downstream disease-altered stress granule pathways. Finally, understanding the molecular triggers that lead to pheno-conversion may lead to opportunities that can delay symptomatic disease onset. A large body of evidence implicates RAN-translated DPRs as a main driver of C9-ALS/FTD. Promising therapeutic strategies for these devastating diseases are being rapidly developed with several approaches already in or approaching clinical trials.

Repeat-associated non-ATG (RAN) proteins have been reported in 11 microsatellite expansion disorders but the factors that allow RAN translation to occur and the effects of different repeat motifs and alternative AUG-like initiation codons are unclear. We studied the mechanisms of RAN translation across myotonic dystrophy type 2 (DM2) expansion transcripts with (CCUG) or without (CAGG) efficient alternative AUG-like codons. To better understand how DM2 LPAC and QAGR RAN proteins are expressed, we generated a series of CRISPR/Cas9-edited HEK293T cell lines. We show that LPAC and QAGR RAN protein levels are reduced in protein kinase R (PKR)(-/-) and PKR-like endoplasmic reticulum kinase (PERK)(-/-) cells, with more substantial reductions of CAGG-encoded QAGR in PKR-/- cells. Experiments using mutant eIF2 alpha-S51A HEK293T cells show that p-eIF2 alpha is required for QAGR production. In contrast, LPAC levels were only partially reduced in these cells, suggesting that both non-AUG and close-cognate initiation occur across CCUG RNA5. Overexpression of the alternative initiation factor eIF2A increases LPAC and QAGR protein levels but, notably, has a much larger effect on QAGR expressed from CAGG-expansion RNA5 that lack efficient close-cognate codons. The effects of eIF2A on increasing LPAC are consistent with previous reports that eIF2A affects CUG-initiation translation. The observation that eIF2A also increases QAGR proteins is novel because CAGG expansion transcripts do not contain CUG or similarly efficient close-cognate AUG-like codons. For QAGR but not LPAC, the eIF2A-dependent increases are not seen when p-eIF2 alpha is blocked. These data highlight the differential regulation of DM2 RAN proteins and eIF2A as a potential therapeutic target for DM2 and other RAN diseases.

Repeat associated non-AUG (RAN) translation is found in a growing number of microsatellite expansion diseases, but the mechanisms remain unclear. We show that RAN translation is highly regulated by the double-stranded RNA-dependent protein kinase (PKR). In cells, structured CAG, CCUG, CAGG, and G(4)C(2) expansion RNAs activate PKR, which leads to increased levels of multiple RAN proteins. Blocking PKR using PKR-K296R, the TAR RNA binding protein or PKR-KO cells, reduces RAN protein levels. p-PKR is elevated in C9orf72 ALS/FTD human and mouse brains, and inhibiting PKR in C9orf72 BAC transgenic mice using AAV-PKR-K296R or the Food and Drug Administration (FDA)-approved drug metformin, decreases RAN proteins, and improves behavior and pathology. In summary, targeting PKR, including by use of metformin, is a promising therapeutic approach for C9orf72 ALS/FTD and other expansion diseases.

More than 40 different neurological diseases are caused by microsatellite repeat expansions. Since the discovery of repeat-associated non-AUG (RAN) translation by Zu et al. in 2011, nine expansion disorders have been identified as RAN-positive diseases. RAN proteins are translated from different types of nucleotide repeat expansions and can be produced from both sense and antisense transcripts. In some diseases, RAN proteins have been shown to accumulate in affected brain regions. Here we review the pathological and molecular aspects associated with RAN protein accumulation for each particular disorder, the correlation between disease pathology and the available in vivo models and the common aspects shared by some of the newly discovered RAN proteins.